A. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions useful in modulating or inhibiting human telomerase activity. In certain embodiments, the invention concerns the use of these agents in treatment of proliferative cell disorders, particularly for cancers whose proliferation is determined by processive telomerase activity.
B. Description of Related Art
Telomeres play an important role in chromosome organization and stability. Human telomerase is a terminal transferase that adds TTAGGG units onto the telomere end. In general, telomerase activity is not detected in normal somatic cells leading to the implication of telomerase in cancer and the impetus to develop agents that selectively target telomerase activity.
1. Genomic Instability of Cancer Cells
One of the general characteristics of cancer cells is genomic instability. Though it is still unclear what causes this instability, a hypothesis gaining increasing attention is that free chromosome ends, either from chromosome breakage or from loss of the telomere sequences which cap the ends, are prone to illegitimate recombination events. Thus, telomeres provide stability to the chromosomes. However, there appears to be a gradual loss of telomere sequences with each cell division, perhaps because of the end-replication problem. Tumor cells have shortened telomeres, but they also possess greatly elevated levels of the enzyme telomerase to overcome the end-replication problem, while normal cells do not. Thus, telomerase is an attractive-target for new anti-cancer agents because of the expected selectivity for neoplastic cells.
2. Telomeres
Telomeres consist of simple DNA repeats at the end of eukaryotic chromosomes and the proteins that bind specifically to those sequences in whole cells (Blackburn, 1991; Zakian, 1989). Telomeric DNA sequences and structures are conserved among widely divergent eukaryotes. The essential telomeric DNA consists of a stretch of a G-rich tandemly repeated sequence. Human and other vertebrate telomeres are based on TTAGGG repeat units. The telomere provides a protective xe2x80x9ccapxe2x80x9d for the end of the chromosome. Broken chromosomes and free DNA ends are susceptible to end-to-end fusion leading to dicentric, ring or other unstable chromosome forms, and to exonucleolytic degradation (Haber, 1984; Mann, 1983; McClintock, 1941; McClintock, 1942; Roth, 1988). By protecting against these events, telomeres prevent loss of genetic information from sub-telomeric regions of the chromosome.
Telomeres, the ends of eukaryotic chromosomes, are composed of tandemly repeated guanine-rich sequences which have an important role in chromosome organization and stability. However, due to the nature of DNA synthesis, the 5xe2x80x2 ends of telomeres shorten with each round of replication leaving a 3xe2x80x2 overhang that is subject to degradation. This has been described as the xe2x80x9cend-replicationxe2x80x9d problem of linear chromosomes (Watson, 1972; Olovnikov, 1973). The end-replication problem can be overcome by addition of nucleotides to the 3xe2x80x2 end of the telomere. A telomere terminal transferase (telomerase) activity was initially discovered in Tetrahymena (Greider and Backburn, 1985). Telomerase activity has since been found in other ciliates (Zahler and Prescott, 1988; Shippen-Lentz and Blackburn, 1989), Xenopus (Mantell and Greider, 1994), yeast (Cohn and Blackburn, 1995), mouse (Prowse et al., 1993), and human cells (Morin, 1989). Telomerase is a ribonucleoprotein in which the internal RNA component serves as a template for directing the appropriate telomeric sequences onto the 3xe2x80x2 end of a telomeric primer. The cloning (Greider and Blackburn, 1989) and secondary structure determinations of the Tetrahymena telomerase RNA have determined the template portion of the RNA which has suggested a model for the mechanism of telomerase activity. Telomerase is thought to act by: 1) Telomerase binding to the 3xe2x80x2 single-stranded overhang of the telomere (TTAGGG in humans) which base pairs with the complementary bases of the RNA component of telomerase, 2) Nucleotide addition onto the 3xe2x80x2 end of the telomere by telomerase using its RNA component as a template, and 3) Dissociation of the newly synthesized telomeric DNA from the RNA template and repositioning to allow for the next round of polymerization. This last step is called the translocation step.
The variety of secondary structures formed by the guanine-rich telomeric sequences involving G-quartets or hairpins(Guschlbauer, 1990; Williamson, 1994) may have an affect on telomerase activity. For example, there is evidence that the G-tetraplex structures formed by telomeric sequences may hinder initial telomerase binding (Zahler et al., 1991). On the other hand, it has been proposed that G-tetraplex formation may actually facilitate the translocation step.
G-quartet structures may also have a role in telomere function. For example, it has been shown that a variety of proteins will preferentially bind to G-quartet structures (Williamson, 1994). Also, the interaction between guanine-rich DNA strands may be involved in the association of chromosomes seen in cells in the presence of varying concentrations of Na+ (Diaz and Lewis, 1975). The function of chromosomal association is unknown but it has been proposed that it is important in such functions as homologous pairing involved in meiosis (Sen and Gilbert, 1988). Recently, a yeast nuclease (Kem1p) was found to specifically recognize and cut only G-quartet structures (Liu and Gilbert, 1994). Deletion of this enzyme was shown to cause telomere shortening, cellular senescence, and blockage in the pachytene stage of meiosis in yeast (Bahler et al., 1994; Tishkoff et al., 1995; Liu et al., 1995).
Another possible function of telomeres has sparked a great deal of interest in cancer research. It has been recently proposed that telomere length may serve as a xe2x80x9cmitotic clockxe2x80x9d (Harley, 1995; Shay, 1995). Normal cells in which telomeres shorten to a critical length become senescent (Allsopp et al., 1992; Harley, 1991). In contrast, immortal cancer cells have an unlimited replicative capacity. Due to the findings that telomerase activity is present in a variety of tumor cells, (Chadeneau et al., 1995; Counter et al., 1994; Counter et al., 1995; Kim et al., 1994) it appears that activation of telomerase is one link to cellular immortality. This makes inhibition of telomerase an ideal strategy for anti-cancer therapy. A number of nucleoside reverse transcriptase inhibitors show anti-telomerase activity in human and Tetrahymena (Strahl and Blackburn, 1994; Strahl and Blackburn, 1996).
3. Chromosome End Replication Problem
Telomeres play a critical role in allowing the end of the linear chromosomal DNA to be replicated completely without the loss of terminal bases at the 5xe2x80x2-end of each strand. Watson (1972) and Olovnikov (1971, 1973) independently described the xe2x80x9cend-replicationxe2x80x9d problem, i.e., the inability of DNA polymerase to replicate fully the ends of a linear DNA molecule. All known DNA polymerases require a primer to initiate polymerization that proceeds in 5xe2x80x2xe2x86x923xe2x80x2 direction. After degradation of the RNA primers, filling-in of internal gaps, and ligation events, the parental strand remains incompletely copied. Thus, in the absence of mechanisms to overcome the end-replication problem, the 5xe2x80x2 end of the newly synthesized DNA in each duplex is shortened following every round of DNA replication. The 3xe2x80x2 single stranded overhang, if not degraded, is converted to a double stranded deletion in the subsequent generation.
4. Telomerase
Immortal cells appear to overcome the end-replication problem by using telomerase to add telomeric DNA repeats to chromosomal ends. Because its mechanism of action involves the copying of an RNA template into DNA, telomerase can be classified as a reverse transcriptase. However, unlike typical reverse transcriptases from retroviruses or lower eukaryotes, it is a ribonucleoprotein that contains its own RNA template as an integral part of the enzyme (Blackburn, 1992). The RNA moiety of telomerase from various ciliates has been cloned and sequenced. For example, the Tetrahymena telomerase RNA moiety is a 159-nucleotide RNA in which a 3xe2x80x2-CAACCCCAA-5xe2x80x2 (SEQ ID NO:1) sequence serves as the template for the synthesis of TTGGGG repeats (Greider, 1989). In Euplotes telomerase, a 15 nucleotide portion, 3xe2x80x2-CAAAACCCCAAAACC-5xe2x80x2 (SEQ ID NO:2) of a 191 nucleotide RNA was found that could serve as a template for the synthesis of TTTTGGGG repeats (Shippen-Lentz, 1990). It is of interest to note that the Euplotes 191 nucleotide RNA and the Tetrahymena 159 nucleotide. RNA share little overall primary sequence similarity. However, despite their divergent primary structures, the Euplotes 191-nucleotide RNA, the telomerase RNAs from T. thermophila, and these of other ciliates can all be folded into similar secondary structures with the putative telomeric template domains for each RNA lying in a corresponding position (Shippen-Lentz, 1989). Studies indicate that human telomerase also contains an endogenous RNA as template (Morin, 1989). The RNA component of human telomerase has been cloned (Feng, 1995).
Telomerase activity from human cells possesses a number of characteristics. First, in the presence of a G-rich human telomeric primer (TTAGGG)3 (SEQ ID NO:3), TTP, dATP and [xcex1-32 P]dGTP, a ladder consisting of bands spaced six bases apart will form (Morin, 1989). Second, since telomerase contains an RNA component, telomerase activity can be obliterated in the presence of RNase A. It has been shown that the bands formed in the presence of excess cold nucleotides TTP and dATP and limiting amounts of [xcex1-32P]dGTP (1.56 xcexcM) are indicative of a pause site at the first guanine in the repeating unit of TTAGGG (Morin, 1989).
Evidence to date indicates telomerase is present in tumor cells but not in normal somatic cells. Thus, telomerase is an attractive novel drug target because there is a strong possibility for selectivity. Strahl and Blackburn (1994) has reported that several chain-terminating inhibitors (arabinofuranosyl-guanine triphosphate, Ara-GTP and 2xe2x80x2, 3xe2x80x2-dideoxyribofuranosyl guanine triphosphate, ddGTP) efficiently inhibit Tetrahymena and human telomerase (Strahl and Blackburn, 1996).
5. Telomere/Telomerase in Cellular Senescence, Immortalization and Cancer
Early studies on human chromosome ends demonstrated that somatic (peripheral blood) telomeres appeared significantly shorter than germline (sperm) telomeres from the same individual (Cooke, 1986; Allshire, 1988; de Lange, 1990). It is now generally known that in most (if not all) somatic tissues, chromosomes gradually lose their terminal telomere sequence with each cell division (Harley, 1990; Hastie, 1990; Lindsey, 1991; Allsopp, 1992; Shay, 1993; Vaziri, 1993; Klingelhutz, 1994). In contrast, sperm telomeres increase in length with donor age, indicating that telomeres are actively maintained and even elongated in the germline (Allsopp, 1992). One explanation for this difference is that telomerase is active in germline cells but somehow is turned off in normal somatic tissues. This hypothesis has some support as no detectable telomerase activity has been found in extracts of embryonic kidney cells, or normal ovarian epithelium (Counter, 1992; Counter, 1994).
Whether telomere shortening has an impact on the proliferative activity of somatic cells remains unknown. The minimum telomere length required for maintaining full telomere function has not been fully established. Some evidence suggesting that telomere shortening could play a role in cellular aging comes from the analysis of primary human fibroblasts grown in culture (Harley, 1990; Allsopp, 1992). These cells lose approximately 50 bp per doubling and eventually stop dividing at a senescence stage call M1. Cells arrested at the M1 stage can be rescued by a variety of viral agents (Counter, 1992; Ide, 1984; Wright, 1989; Radna, 1989). The virally transformed cells continue to divide for as many as 50 cell divisions before they face another crisis, called M2, which is characterized by a balance of cell divisions and cell death (Counter, 1992). Cells that have bypassed the M1 arrest continue to lose.their telomeric DNA, resulting in much shorter telomeres. An average telomere length of approximately 1.5 kbp or less as the cell approaches M2 may not contain sufficient telomere sequence to sustain normal telomere function.
Occasionally, virally transformed cultures yield immortal cells that have overcome the M2 crisis. Interestingly, unlike the senescent cells, the telomere of the immortal cell lines is stabilized by re-activation of the enzyme telomerase. Counter has postulated that telomerase activation is an obligatory step in the immortalization of human cells (Counter, 1992). In support of this hypothesis, telomerase activity was recently detected in metastatic human ovarian carcinoma cells but not in normal control cells, including healthy ovarian epithelium (Counter, 1994).
6 Telomerase Biochemistry
Alternatively, nucleotide analogs that are incorporated into telomeres by the action of telomerase may interfere with the function of the telomeres. For example, some nucleotide analogs so incorporated may block the ability of telomeres to form G-quarted or G-hairpin structures, thereby rendering the telomeres unable to be recognized by protein which specifically bind these structures. In the current model for the mechanism of telomeric DNA synthesis by telomerase (Blackburn, 1990), the 3xe2x80x2 nucleotides of the overhang region of the chromosome terminus base-pair with a telomere-complementary sequence in the telomerase RNA. Next, the chromosomal end is extended using the RNA as a template, resulting in the addition of six telomeric nucleotides. Then, the extended DNA terminus unpairs from its RNA template and is repositioned on the 3xe2x80x2 portion of the template, becoming available for another round of elongation by telomerase.
One of the most striking features of the telomerase reaction is that it involves not only copying of an internal template, but also an efficient translocation event which occurs after the last [5xe2x80x2 most] residue of the template has been copied into DNA. The translocation step has been deduced from the processive nature of the telomerase reaction in a cell-free assay. Thus, telomerase initiates synthesis on a telomeric sequence DNA primer, and in the presence of an excess of the same primer or of a high concentration of a challenging primer, continues to elongate the first primer up to hundreds of nucleotides before dissociation (Blackburn, 1992).
Not all telomerase preparations are processive in the cell-free assay. Nonprocessive telomerase activity has been described in mouse FM3A cells (Prowse, 1993), Tetrahymena (Collins, 1993) and Xenopus (Mantell, 1994). Both processive and nonprocessive telomerases have been identified by the inventors from different cell lines. Intriguingly, the telomerase in S100 extracts of the human HeLa-S3 subline is non-processive, while the telomerase in the parental HeLa cells is processive. Whether these are two different enzymes or the same enzyme with different isoforms is currently being investigated. Whether processivity or non-processivity of the activity identified in the biochemical assay is relevant to telomerase function in whole cells remains to be elucidated. Evidence suggesting that telomerase functions nonprocessively in whole cells has been documented (Blackburn, 1992).
7 Deficiencies in the Prior Art
Strahl and Blackburn (1994) have tested AZT-TP, ara-GTP and ddGTP against a non-mammalian Tetrahymena telomerase and human telomerase (Strahl and Blackburn, 1996). Detailed methods and agents for inhibiting telomerase have not been described. In addition, there has been no distinction made between the telomerase produced in some normal cells and the telomerase produced by cancer cell. The identification of therapeutic compounds which have modulation or inhibitory activity against human telomerase is a desirable goal, particularly to identify compounds and develop methods of treatment of cancers in which processive telomerase contributes to the immortality and undesirable proliferation.
Toward this end and because of the important if not entirely understood role of telomerase in cell growth and senescence, there has been an effort to identify compounds that affect telomerase activity. Use of such compounds in controlling cell proliferation has obvious implications in treatment of malignant cancers. A goal of current medical investigation is to understand and treat cellular disorders, preferably to selectively target cancer cells either by altering the telomere, the telomerase, or the enzyme structure and/or by inhibiting telomerase.
The present invention overcomes one or more of these and other drawbacks inherent in the prior art by providing compositions and methods for their use in the inhibition and modulation of eukaryotic telomerase activity. More particularly, certain compositions have been shown to modify telomerase activity in cancer cells to more nearly approximate that found in normal or benign cells. Using a cell free biochemical assay, several classes of compounds, including nucleoside triphosphates and their derivatives have been identified as having an inhibitory effect on human telomerase. These compounds have a surprising effect on telomerase in apparently modulating processive telomerase to a non-processive activity. These results now offer new avenues of therapy for treatment of cancers characterized by cells with processive telomerase activity.
In the quest for finding nucleotide analogues that inhibit telomerase, 7-deaza-2xe2x80x2-deoxyguanosine triphosphate and 7-deaza-2xe2x80x2-deoxyadenosine triphosphate were found to be particularly potent inhibitors of telomerase activity. These compounds were originally investigated based on the rationale that the N-7 nitrogen of purine bases is required for Hoogsteen base-pairing involved in secondary structures formed by telomeric sequences. Unexpectedly, the 7-deaza nucleotides turned out to be poor substrates for human telomerase and were effective modulators of processive telomerase. The nucleotides thus not only present a novel mode of telomerase inhibition but also are useful for the study of the role of DNA secondary structure in telomerase mechanism.
The compounds of the present invention have the general structure (A), (B) or (C) 
where U is independently carbon or nitrogen; R1 is independently H, lower alkyl, or phenyl alkyl; R is independently H, lower alkyl, NH2, halogen, azido or alkene; P is independently oxygen or sulfur; and S is an acyclic or cyclic glycosyl group represented by the formulae: 
where R3 is independently H, halogen, amino, azido, or hydroxyl; R4 is independently H or OH; V is oxygen or methylene; and R5 is OH, (CH2)n PO(OR7)2, OP(O)(OR7)2; R6 is H, lower alkyl, hydroxy-substituted lower alkyl, or haloalkyl; R7 is H, lower alkyl, CH2OCO(branched or straight chain alkyl (C1-C8) or aryl, and R8 is H or CO(lower alkyl) and n is 1-2; 
where K is oxygen or methylene; G is independently oxygen, sulfur or methyl; F is independently oxygen or sulfur; and the glycosyl bond between S and U is xcex1 or xcex2.
The 7-deaza compositions of the present invention found to be useful telomerase-inhibiting analogs may be modified in any of several ways. It is convenient to consider the design of analogs and derivatives in three general categories: (1) modifications of the heterobase of the nucleoside triphosphate. (2) modifications of the ribose sugar; and (3) modifications of the phosphate backbone.
The discovery that 7-deaza-2xe2x80x2-deoxynucleosides are effective in altering telomerase activity, has led to the conclusion that there are a wide range of modifications that can be made in this general class of deaza purines and pyrimidine nucleosides thus providing a plethora of compounds for selection by the practitioner in seeking to modulate telomerase activity.
Particular structures of the base portion of the nucleotide compounds contemplated by the inventors as useful for telomerase modulation or inhibition include the guanine analogs shown:
Deaza-Guanines or Aza-Guanines, 6-Thioguanine 
These compounds are summarized in the generalized formula below: 
The base components of dATP, dGTP, and dTTP are Adenine, Guanine, and Thymine, respectively. The structure of adenine, guanine, and thymine is shown below: 
Likewise, one may use adenines, such as deaza or azoadenines: 
In like manner modified thymine bases may be used, including: 
The following general formula summarizes these modifications on the thymine base. 
The bond between the base and the sugar moiety may be alpha or beta. The natural nucleoside is the xcex2-D form. Nonnaturally occurring forms include xcex2-L, xcex1-D and xcex1-L. 
Further examples of nucleotide analogs with sugar modification include the 2xe2x80x2-Deoxy-3xe2x80x2-deoxy-3xe2x80x2-substituted nucleosides (or nucleotides): 
Of course the sugar moiety need not be limited to any particular sugar and several sugars are contemplated as suitable including in addition to 2xe2x80x2-deoxyribose, ribose, arabinose, xylose, and lyxose 
Carbocyclic compounds may be substituted for the natural sugars: 
Acyclic compounds would also be expected to substitute for the sugar residue. 
The following modified phosphate groups may be attached to appropriate purine or pyrimidine nucleosides, particularly to 7-deaza-2xe2x80x2-deoxyadenosine and 7-deaza-2xe2x80x2-deoxyguanosine residues.
Particularly preferred 7-deaza compounds useful for the practice of the present invention are 7-deaza-dGTP and 7-deaza-dATP, having the structures shown: 
These analogs have been shown to affect telomerase activity as follows: (i) Both compounds inhibit telomerase in a dose-dependent manner; (ii) 7-deaza-dGTP and 7-deaza-dATP are incorporated into telomeric DNA by telomerase. 7-deaza-dATP can promote non-progressive activity by telomerase. However, incorporation of 7-deaza-dATP or 7-deaza-dGTP results in a telomeric ladder that is prematurely shortened; and (iii) Substrate inhibition (or allosteric inhibition) of 7-deaza-dGTP or 7-deaza-dATP is observed at a high concentration. 
Synthetic procedures for the preparation of nucleotide analogs are available and some of these are preparable by analogy to procedures found in the literature (Scheit, 1980). A potential general synthetic pathway is outlined below for 7-substituted-7-deaza-dGTP xcex1-phosphorothioates. Selected 7-substituted-7-deaza-dATP xcex1-phosphorothioates could be prepared in a similar manner. 
Where Q is a protecting group such as a silyl protecting group, e.g. tet-butyl-dimethyl silyl, tetrahydropyranyl, benzyl, etc. In further aspects of the invention it has been found that human telomerase contains a 3xe2x80x2-5xe2x80x2 exonuclease activity, analogous to Tetrahymena telomerase. Thus the synthesis of human telomeric DNA by telomerase is a balance of the 3xe2x80x2-5xe2x80x2 exonuclease activity and the 5xe2x80x2-3xe2x80x2 polymerase activity. In principle, synthesis of telomeric DNA can be reduced by blocking the polymerase or by stimulating the exonuclease, or both. One should be able to stimulate the exonuclease by drug interaction, thereby providing an effective method to shorten telomeres rapidly in cancer cells that produce high levels of telomerase, leading to a rapid cell death. Methods for stimulating the exonuclease include altering the interaction of dTTP and possibly other nucleotides with the telomerase. Compounds that block or limit dTTP are contemplated as useful with such a method.
Telomerase has a proof-reading-like exonuclease activity, so that incorporated modified nucleotides can be removed with no effect. The inventors contemplate that excision of the modified nucleotides can be achieved by blocking the telomerase exonuclease with an alpha-thionucleotide. Such incorporated nucleotides, cannot easily be removed by exonuclease activity because the thio-phosphate linkage is not cleaved by exonucleases. Suitable compounds include alpha, beta and gamma thio 7-deaza guanosine, adenosine and thymidine triphosphates such as for example alpha-thio-7-deaza-dGTP.
Some of these nucleotide analogs appear to also inhibit telomerase polymerization by a competition or allosteric mechanism mediated through telomerase inhibition.
The inventors believe that 7-deaza-2xe2x80x2deoxynucleoside purines and pyrimidines herein described as telomerase modulators or inhibitors will be of particular use in the treatment of human cancers associated with high levels of processive telomerase. However, in order for these 2xe2x80x2-deoxynucleosides to exert their activity, they must be converted to triphosphates intracellularly, i.e., 7-deaza-2xe2x80x2-deoxyguanosine must be transported into the cells and be phosphorylated by a nucleoside kinase to 7-deaza-dGMP and, subsequently, further phosphorylated to 7-deaza-dGDP and 7-deaza-dGTP, respectively. 
In general, phosphorylation of a nucleoside to its monophosphate is a rate-limiting step in whole cells. If the cells were unable to phosphorylate the nucleosides to monophosphates, no intracellular di- or triphosphates can be formed or identified. On the other hand, one also cannot simply incubate the cells with the phosphates (mono-, di-, or triphosphates) and expect them to transport into the cells and provide the cells with the triphosphates of nucleoside analogs. The phosphates are highly negatively charged, and therefore, will not transport into cells. Instead, the mono-, di-, or triphosphates will be dephosphorylated extracellularly by alkaline phosphorylase or 5xe2x80x2-nucleotidase back to the nucleoside.
In order to circumvent these problems, one can prepare prodrugs of monophosphates in which the negatively charged phosphate group is functionalized. The prodrug of monophosphates will then contain no charged groups and can be efficiently transported into cells. Once it has entered the cells, the protective group is hydrolyzed by esterase, and nucleoside monophosphate is released. Subsequently, phosphorylation of the liberated monophosphate will produce the desired nucleoside triphosphate. The general structure of the prodrugs is shown below: 
The use of this type of prodrug approach to deliver nucleotides to cells has been described (Farquhar et al., 1983; Sastry et al., 1992). Such compounds can enter cells and release the monophosphates which can be further phosphorylated to the corresponding triphosphates.
The present invention has identified one of the most potent telomerase inhibitors, 7-deaza-2xe2x80x2-deoxyguanosine-5xe2x80x2-triphosphate. The IC50 value (6.8 xcexcM) is at least 50-times more potent than AZT-TP. 7-deaza-2xe2x80x2-deoxyguanosine-5xe2x80x2-triphosphate lacks the 7-nitrogen atom which is essential for the formation of G-quartets or hairpins. If 7-deaza-2xe2x80x2-deoxyguanosine-5xe2x80x2-triphosphate is incorporated into telomeres, further telomere elongation may be prevented.
Several nucleoside triphosphate analogs with phosphate backbone modifications were demonstrated to be potent telomerase inhibitors. These phosphate backbone modifications include (1) thiophosphate, where the (xcex1) Pxe2x95x90O bond is replaced with a Pxe2x95x90S bond, e.g., NAO13 (IC50=81.5 xcexcM) and (2) phosphonate, where the oxygen atom joining the xcex2 and xcex3 phosphate (P) atom is replaced with a methylene (xe2x80x94CH2xe2x80x94) group e.g. NA014 (IC50=102 xcexcM). (One of the most potent inhibitors identified was 7-deaza-2xe2x80x2-deoxyguanosine-5xe2x80x2-triphosphate (NA022) with IC50 value of 6.8 xcexcM. 7-deaza-2xe2x80x2-deoxyadenosine-5xe2x80x2-triphosphate (NA023) also inhibits telomerase activity with an IC50 value of 78.5 xcexcM);
It is now possible to define the structure-activity relationship of nucleoside/nucleotide analogs as inhibitors and modulators of telomerase, thus allowing additional specific inhibitors and modulators of the enzyme to be identified. The design and synthesis of potent telomerase inhibitors based on these studies provide an array of telomerase modulating drugs to be used in the treatment of proliferative cell disorders, and particularly those involving cancers characterized by high levels of processive telomerase.
Useful telomerase-inhibitory compounds are not believed to be limited in any way to the specific compounds or nucleotide analogs and derivatives specifically disclosed herein. In fact, it may prove to be the case that the most useful pharmacological compounds designed and synthesized in light of this disclosure will be second generation derivatives or further-chemically-modified compositions.
Where telomerase-containing cells are located within an animal, a pharmaceutically acceptable composition of the telomerase inhibitor may be administered to the animal in an amount effective to modify the telomerase activity of the target cell. In terms of inhibiting telomerase activity in tumor cells, this is contemplated to be an effective mechanism by which to treat cancer that will have very limited side effects.
An embodiment in which the compositions of the present invention find particular utility is the treatment of cell proliferative disorders, and in particular human tumors characterized as having processive telomerase. The utilization of telomerase inhibitors (which either directly inhibit the telomerase activity or indirectly incorporate into telomere and thus prevent telomere further elongation) will lead to progressive telomere shortening in tumors where telomerase is active. Once the telomere length shortens to a critical length (ca 2 kb), the tumor will go into crisis and eventually die. These telomerase inhibitors will have little or no effect on the normal somatic cells because telomerase activity in normal cells is generally low or undetectable.
It is believed that the disclosed compounds will be useful for potentially treating a patient after surgical removal of a tumor. The patient would be treated with non-cytotoxic doses of nucleoside/nucleotide analogs for a prolonged period of time to prevent the recurrence of micro-metastasis. Alternatively, effective treatment of invading pathogens susceptible to telomerase inhibition are also contemplated, as are applications in treating age-related disorders such as atherosclerosis and osteoporosis.
There are several methods contemplated by the inventors for the delivery of such telomerase inhibitors into cells. In one embodiment, cells are provided with the corresponding nucleoside analogs, and subsequent cellular metabolism converts the nucleosides into nucleoside mono-, di- and tri-phosphates. In another embodiment, formulations of specific telomerase inhibitors are prepared in vehicles which protect the nucleotide from phosphatase degradation and facilitate the transport of nucleotides. In the case of the latter, a preferred method for delivery would be that of liposome-mediated delivery. In yet another embodiment, one could prepare pro.
The therapeutic potential for liposome-mediated transfer of such telomerase inhibitors into human cells is well known to those of skill in the art. Based on existing evidence which shows that the systemic injection of cationic liposome complexes into animals is non-toxic (Stewart et al., 1992), the inventors contemplate the use of such liposome-mediated methods for introducing the compositions disclosed herein into animal subjects.
Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Chang and Brenner, 1988; Muller et al., 1990). Liposomes have been used effectively to introduce drugs (Heath et al., 1986; Storm et al., 1988; Balazsovits et al., 1989), radiotherapeutic agents (Pikul et al., 1987), and enzymes (Imaizumi et al., 1990; Imaizumi et al., 1990) into a variety of cultured cell lines and animals. In addition, several successful clinical trails examining the effectiveness of liposome-mediated drug delivery have been completed (Lopez-Berestein et al., 1985; Coune, 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery Nabel et al., 1992; Mori and Fukatsu, 1992).
Introduction of the liposome-telomerase inhibitor complex may be by injection, either systemically into peripheral arteries or veins (including the carotid or jugular vessels), or directly into specific tissues to be targeted. Such liposome formulations are commercially available, e.g., 1:1 (w:w) mixture of the cationic lipid n-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) may readily be employed for such liposome formulations.
Based on the inventors"" discovery of a method to modify or inhibit telomerase activity, it is contemplated that several classes of compounds will be useful. It will be desirable to determine which analogs and derivatives will be most suitable for particular treatments; for example, depending on the type of cancer cell present and particularly the amount and activity of telomerase present. A related aspect of the invention is the discovery that certain allosteric interactive agents will alter or inhibit telomerase activity. Nucleotides such as 7-deaza dGTP and 7-deaza dATP, are capable of allosterically inducing a reduction in telomerase polymerizing activity. The inventors have demonstrated that other nucleotides such as dGTP will not induce this effect, indicating the importance of the 7-deaza modification. This now provides a new method of modulating telomerase for the treatment of cancer by designing a series of 7-deaza compounds that will allosterically bind with telomerase with varying effects on modulating telomerase activity. Highly processive telomerase for example may require stronger inhibitors or modifications to the nucleotide analog that change allosteric interactions or are less efficiently incorporated into the telomere.